Project Summary/Abstract: Chimeric antigen receptors (CARs) are engineered recombinant receptors composed of key signaling modules from both the T cell receptor (TCR) and co-stimulatory receptors to mount effective anti-tumor immunity. Engineered CAR-T cells be reinfused into the patient to recognize and attack cancer cells. CAR-T cell therapy has shown very promising results in clinical trials. However, owing to a lack of precise control of the dose, location, and timing of T cell activity, this method involves some significant safety challenges to be overcome. For example, cytokine release syndrome (CRS), occurred due to a large and uncontrolled release of cytokines in response to CAR-T, may cause symptoms ranging from fever to potentially fatal organ destructions. In addition, conventional CAR T cells are also associated with the targeted destruction of normal tissue, which is known as ?on-target, off-tumor? effects since tumor antigens are also expressed at a certain level in several normal tissues. Therefore, the uncontrolled aggressively amplified CAR-T cells cross-react with cells in the heart, lung or liver and cause devastating consequences in patients. To address this challenging issue, we propose to develop non-invasive methodologies that allow for the spatiotemporal control of chimeric antigen receptor (CAR) T cells for cancer treatment by using B cell lymphoma as a test case. This proposal is based on two key discoveries made by the members of the team. First, we have created a way to optogenetically modulate the function of calcium signaling by conferring visible light sensitivity to stromal interaction molecule 1 (STIM1), activating the ORAI1 calcium channel in T cells to mount effector immune responses. The second is our development of upconversion nanoparticles (UCNPs), more specifically their use as in vivo relay nodes to capture and convert low power, deep tissue-penetrant, and near infrared radiation (NIR) into visible light for in vivo optogentic applications. In our preliminary results, we have demonstrated that the use of ex vivo UCNPs and optogentic dual engineering approach can indeed optogenetically instruct immune cells (i.e,, dentritic cells) to attack melanoma in mice, and that NIR light therapy effectively suppresses melanoma growth and metastasis to lungs. We propose three specific aims. For Aim 1, we will develop photo-tunable CARs in engineered therapeutic T cells. In Aim 2, we will devise new generations of UCNPs with improved compatibility with OptoCARs. In Aim 3, we will determine the efficacy of nano-optogenetic CAR-T platforms in vitro and in vivo. This new strategy will overcome many of the limitations of current CAR-T based approaches, and will enable new applications in both fundamental science and human health.